1310406 玖、發明說明' 【發明所屬之技術領域】 本發明大體上係關於測定核酸被測物的方法及儀器。 尤其，本發明是關於偵測一不含傳統光學可辨識標誌（標 記）物質的被測物。 【先前技術】 使用一實質上爲平面的系統，專家稱之爲生物感應器 或生物晶片，以測定特定核酸被測物之性質與/或含量，例 如DNA，是所熟知的。這些生物晶片的表面含有撐體，通 常會形成許多偵測區域，大多的例子是排列成格子狀，此 處每一個別區域或一群區域對特定待測之被測物具有的專 一性都彼此不同。在待測DNA被測物的例子中，其專一的 核酸探針，例如，寡核苷酸或cDNA，大多是形成單股的形 式，且其對核酸的個別專一性是由序列順序所決定（探針 設計），以直接或間接固定於撐體表面的個別區域。在本 文相對應的偵測方法，將以此種方式作用的晶片表面與欲 測定的DNA被測物接觸，在此狀況下確定（確定先前可偵 測的已標記之標的核酸是存在的結果下）後者與固定探針分 子雜交。稍後對一個或更多專一形成的雜交複合物進行定 性與視需要的定量偵測，大多數例子係利用物理光學冷光 偵測，及分配所得到的數據到個別的偵測區域，因此可以 測定，例如，核酸被測物的存在或序列及視需要的其含量 〇 除了這些以冷光爲基礎的方法，在這幾年的努力下， 1310406 DNA的分析不需要冷光標定且不需要利用偵測及影像。 舉例來說，嘗試利用場效電晶體以區別單股及雙股時 期的狀態（E. Souteyrand等人，藉場效應直接偵測合成的 同形寡聚物DNA序列之雜交（Direct detection of the hybridisation of synthetic homo-oligomer DNA sequences by field effect) 5 /. Phys. Chem. B. ' 1001，2980，[1997])或利 用阻抗結構（見，例如，P. VanGerwen等人，奈米級交叉 指型電極排列做爲生化感應器（Nanoscaled interdigital electrode arrays for biochemical sensors)，Sensors and Actuators，B49，73-80，[1998])。 該技藝的另一方法是有關於利用沙雷氏桿菌（&/7^仏 )細胞外內切酶的酵素活性，其中酵素媒介的 DNA分解導致酸鹼値的改變，可被酸鹼感應器偵測（S· Reher，利用沙雷氏桿菌細胞外內切酶酵素活性以電量法、 電位法及光學法分析DNA及RNA(DNA and RNA analysis by voltammetric, potentiometric and optical methods using the extracellular endonucleases of Serratia marcescens) ， ISBN 3-89825-030-X ， 1999)。 此外，一現有的發表中，描述利用特定的標誌（標記 )物質施行DNA的分析，但其偵測不利用光學方法。一方 法是關於利用電子標記標定雜交的DNA，及讓此雜交DNA 吸附到貴金屬電極上，此處的結合事件可利用電極讀取。 (www.microsensor.com/TechnologySystem.html ， Clinical microsensors，2000 )。其他文獻描述偶合一小的順磁體到 1310406 DNA分子上，藉由磁場的變化來讀取。（D.R. Baselt等人 ’以磁致電阻技術爲基礎的生物感應器（A biosensor based on magnetoresistance technology) ， Biosensors & 万1998，13 (7-8) : 731-9，[1998])。 雖然上述所提到的文獻示範其他不同於以冷光爲基礎 的核酸分析，卻遭遇雜交反應需花數小時時間的困難，加 上用來測量的感應器測量上的偏差，即專家所知的偏移 (drift) 〇此偏移導致訊號隨時間改變，其通常無法從實際的 訊號中被辨識或適當的分離，因後者與此偏移位於同一頻 率強度數量級。進而，通常較容易讀到在最短可能時間內 達到最大値的訊號。 【發明內容】 因此，本發明的任務是提供一改善的方法，以克服與 偏移問題有關的缺點。 此任務根據本發明中主要申請專利範圍的方法而解決。 根據一項具體實例，本發明是有關測定核酸被測物的 方法，其係藉由將被測物雜交至適合的固定在固相的核酸 探針上，其中 (a) 此核酸被測物與核酸探針在適合的雜交狀態下一起 培養，形成雜交複合物，且 (b) 此被測物係基於特別與雜交複合物質量之酵素依賴 性增加或減少相關的物理化學測量數據而測定’ 藉此數據之測量是利用至少一個感應器之方法進行’ 而該感應器是固相中不可缺少的一部分。 1310406 由於這種間接的解決方法，本發明成功的大大避免偏 移問題，其藉由轉換此偵測至不同的時間窗且因此至不同 的頻率，且較佳的只需幾秒鐘或幾分鐘（見圖ο。本方法 較佳的具體實例描述在附屬的申請專利範圍中。 本文中所指的“測定”，是關於分析不只是核酸，尤其 是包含偵測樣品中要檢驗的核酸被測物。進而，包含應用 的形式，例如，測定核酸序列及偵測在特定單核酸多型性 (SNP)中的變異。因此本方法可確保一廣泛的可能使用範 圍，因其可應用至所有目前及未來以雜交複合物形成爲基 礎的測定及/或偵測技術中。 根據一較佳的具體實例，導致雜交複合物質量增加或 減少的酵素是選自由包含聚合酶、連接酶（ligase )、核糖 酶（ribozyme )、準催化性（quasi-catalystic )核酸、去氧 核糖核酸酶（DNase) /核糖核酸酶（RNase)(外及/或內切 核酸酶，包括限制內切核酸酶）及核糖核酸酶H (Rnase Η )所組成的群組中，其中，尤其是具有5’及/或3’-外切核 酸酶活性的聚合酶是更佳的。 除了此DNA依賴的DNA聚合酶外，根據本發明，視 存在的核酸組成（RNA或DNA)而定的質量增加可利用 RNA依賴的DNA聚合酶（反轉錄酶）或RNA依賴的RNA 聚合酶（複製酶）而產生。質量的增加也可利用適當的聚 合酶活化的核糖酶或準催化性的RNA產生。根據本發明， 所有聚合酶（包括核糖酶或準催化性RNA)應用的原則係 熱穩定及不耐熱的酵素都可使用。 1310406 酵素導致的質量增加亦可利用連接酶完成。文中引人 注意處，是一適當的使用本發明提及之連接酶活化之核糖 酶’或準催化性RNA。根據本發明，所有連接酶（包括核 糖酶或準催化性RNA)應用的原則係熱穩定及不耐熱的酵 素都可使用。 相對於質量增加，質量的減少亦可被偵測到。藉由核 酸酶（核糖核酸酶，去氧核糖核酸酶）切除結合的核酸可 導致質量減少。5’及/或3’-外切及內切核酸酶及核糖核酸 酶Η皆可使用。單股及雙股酶或具有兩者活性的酶皆可使 用。關於核酸酶，原則是具有序列專一性及非序列專一性 的酶都可使用。核糖酶或具有核酸酶活性的準催化性RNA 亦可。通常，核糖酶或準催化性RNA其作用具有序列專一 性，此處的專一性，可利用個別的雜交序列的需求調整。 因此，本發明反映了大部分固相結合的核酸被測物所 遭遇的情形，即固定到固相的單股核酸探針的存在（見圖 1Α)。在適合的狀況下，如果核酸被測物與存在的探針序 列互補，即在此探針處形成至少部分至少雙股的雜交複合 物（見圖1Β)。 根據本發明，啓動酶的步驟（見圖1C)是在複合物形 成之後，酶的作用導致一可測得的複合物質量改變。 舉例來說，如果一雜交複合物包含較短的DNA探針， 及存在一相對較長的核酸被測物，在適合的狀況下且含有 四種核苷酸三磷酸（A;T;G;C)，可以使用聚合酶，其 可塡滿（至少部分）由較長的核酸被測物所造成的單股區 1310406 域（見圖1C及ID)。根據假設的平均結合速度，即每分 鐘一仟個鹼基的規模，此連續性的聚合作用在幾分鐘之內 發生。這個例子是假設，此兩個雜交的部分具有不同的長 度，亦可應用在相反的例子中，即被測物的長度較探針爲 短。在這種情況下，有利於將探針設計爲至少有100個核 苷酸的長度，且此雜交複合物的單股區域是可被預期的， 而且其可藉由聚合酶的活性延長而塡滿，使其儘可能靠近 感應器的表面。如果探針是藉由3’端固定的且單股區域塡 滿是在固相發生，亦可達到此優點。這些有益的具體實例 可轉移到不只聚合酶，亦包括一般根據本發明合適的所有 酵素，且容易由習於該項技術的人完成，視其想要應用的 領域而定。 當待測樣品中沒有存在與探針序列互補的被測物時， 則因不同的結合能，故在此時間點中沒有雜交複合物的形 成，因此接下來的酵素反應不會發生，且沒有酵素依賴的 測量數據可被紀錄。 因爲不應用電場的雜交反應通常需耗費數小時，根據 本發明，其偵測的時間相當的減少，導致其可在很短的時 間窗內發生，更適合感應器的讀取。 前文所提作爲例子的聚合酶活性可產生焦磷酸根離子 ，其在雜交複合物單股區域，於核苷酸三磷酸之聚合反應 期間被釋放出來，且導致局部酸化，因而使酸鹼値降低。 藉局部排列pH感應器或pH偵測器（例如，酸鹼感測場效 電晶體元件（PH-ISFET))，若希望可偵測對應特定位置 厂 4«·：1310406 发明, INSTRUCTION DESCRIPTION OF THE INVENTION [Technical Field to Which the Invention Is Alonged] The present invention relates generally to a method and apparatus for measuring a nucleic acid analyte. In particular, the invention relates to detecting a test object that does not contain a conventional optically identifiable marker (marker) substance. [Prior Art] The use of a substantially planar system, known to the expert as a biosensor or biochip, to determine the nature and/or content of a particular nucleic acid analyte, such as DNA, is well known. The surface of these biochips contains a support, and usually many detection areas are formed. Most of the examples are arranged in a lattice shape, where each individual area or group of areas has different specificities for a specific object to be tested. . In the case of the analyte to be tested, a specific nucleic acid probe, such as an oligonucleotide or a cDNA, is mostly formed in a single-strand form, and its individual specificity for nucleic acid is determined by the sequence order ( Probe design) to be fixed directly or indirectly to individual areas of the support surface. In the corresponding detection method herein, the surface of the wafer acting in this manner is contacted with the DNA analyte to be determined, and in this case, it is determined (determining that the previously detectable labeled nucleic acid is present) The latter hybridizes to a fixed probe molecule. Qualitative and as-needed quantitative detection of one or more specifically formed hybrid complexes later, most of which utilize physical optical luminescence detection and assign the resulting data to individual detection areas, thus enabling determination For example, the presence or sequence of nucleic acid analytes and their desirable content eliminates these luminescence-based methods. With the efforts of these years, the analysis of 1310406 DNA does not require cold cursors and does not require detection and image. For example, try to use field-effect transistors to distinguish between single-strand and double-strand periods (E. Souteyrand et al., direct detection of hybridization of synthetic homo-oligomer DNA sequences by field effect (Direct detection of the hybridisation of Synthetic homo-oligomer DNA sequences by field effect) 5 /. Phys. Chem. B. '1001, 2980, [1997]) or using impedance structures (see, for example, P. Van Gerwen et al., nanoscale interdigitated electrodes) Nanoscaled interdigital electrode arrays for biochemical sensors, Sensors and Actuators, B49, 73-80, [1998]). Another method of this technique is to use the enzyme activity of the extracellular endonuclease of Serratia serrata, in which the DNA decomposition of the enzyme medium leads to a change in acid and alkali hydrazine, which can be detected by an acid-base sensor. Detection (S·Reher, DNA and RNA analysis by voltammetric, potentiometric and optical methods using the extracellular endonucleases of Serratia) using the extracellular endonuclease activity of Serratia Marcescens), ISBN 3-89825-030-X, 1999). In addition, in an existing publication, the analysis of DNA using a specific marker (marker) substance is described, but the detection does not utilize an optical method. One method is to calibrate the hybridized DNA using an electronic label, and to adsorb the hybrid DNA to the noble metal electrode, where the binding event can be read by the electrode. (www.microsensor.com/TechnologySystem.html, Clinical microsensors, 2000). Other literature describes coupling a small paramagnetic to a 1310406 DNA molecule, read by changes in the magnetic field. (D.R. Baselt et al. 'A biosensor based on magnetoresistance technology, Biosensors & Wan 1998, 13 (7-8): 731-9, [1998]). Although the above-mentioned literature demonstrates that other nucleic acid analysis based on luminescence is different, it takes several hours for the hybridization reaction to be difficult, plus the deviation in the measurement of the sensor used for measurement, that is, the bias known to the expert. Drift This offset causes the signal to change over time, which typically cannot be identified or properly separated from the actual signal, since the latter is on the same frequency strength level as the offset. Furthermore, it is often easier to read the signal that reaches the maximum chirp in the shortest possible time. SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide an improved method for overcoming the disadvantages associated with the offset problem. This task is solved in accordance with the method of the main patent application scope of the present invention. According to a specific example, the present invention relates to a method for determining a nucleic acid analyte by hybridizing a test substance to a suitable nucleic acid probe immobilized on a solid phase, wherein (a) the nucleic acid analyte is The nucleic acid probes are cultured together in a suitable hybridization state to form a hybridization complex, and (b) the analyte is determined based on physicochemical measurement data particularly related to an enzyme-dependent increase or decrease in the quality of the hybridization complex. The measurement of this data is carried out by means of at least one sensor, which is an indispensable part of the solid phase. 1310406 Due to this indirect solution, the present invention successfully avoids the offset problem by switching this detection to different time windows and thus to different frequencies, and preferably only a few seconds or minutes (See Fig. ο. A preferred embodiment of the method is described in the scope of the accompanying patent application. The term "assay" as used herein refers to the analysis of not only nucleic acids, especially the nucleic acids to be tested in the detection sample. Further, it includes a form of application, for example, measuring a nucleic acid sequence and detecting a variation in a specific single nucleic acid polymorphism (SNP). Therefore, the method ensures a wide range of possible uses since it can be applied to all current And in future assays and/or detection techniques based on hybrid complex formation. According to a preferred embodiment, the enzyme that causes an increase or decrease in the mass of the hybridization complex is selected from the group consisting of a polymerase, a ligase, Ribozyme, quasi-catalystic nucleic acid, deoxyribonuclease (DNase) / ribonuclease (RNase) (external and / or endonuclease, package A group consisting of a restriction endonuclease and a ribonuclease H (Rnase®), among which a polymerase having 5' and/or 3'-exonuclease activity, in particular, is preferred. In addition to this DNA-dependent DNA polymerase, according to the present invention, an increase in mass depending on the nucleic acid composition (RNA or DNA) present may utilize an RNA-dependent DNA polymerase (reverse transcriptase) or an RNA-dependent RNA polymerase (replication) Produced by enzymes. The increase in mass can also be produced using appropriate polymerase-activated ribozymes or quasi-catalytic RNA. According to the invention, the principles of application of all polymerases (including ribozymes or quasi-catalytic RNA) are thermally stable. And heat-labile enzymes can be used. 1310406 Enzyme-induced mass increase can also be accomplished using ligase. It is noted that it is a suitable use of the ligase-activated ribozymes or quasi-catalytic RNAs mentioned in the present invention. According to the present invention, all ligase (including ribozyme or quasi-catalytic RNA) applications are thermally stable and thermolabile enzymes can be used. Compared to mass increase, mass reduction can also be detected. Excision of the bound nucleic acid by nuclease (ribonuclease, deoxyribonuclease) can result in a decrease in mass. Both 5' and/or 3'-exo and endonucleases and ribonuclease can be used. And double-stranded enzymes or enzymes having both activities can be used. For nucleases, the principle is that both enzymes with sequence specificity and non-sequence specificity can be used. Ribozyme or quasi-catalytic RNA with nuclease activity can also be used. In general, ribozymes or quasi-catalytic RNAs have sequence specificity, and the specificity here can be adjusted by the needs of individual hybridization sequences. Therefore, the present invention reflects most of the solid phase bound nucleic acid analytes. The situation encountered, the presence of a single-stranded nucleic acid probe immobilized to the solid phase (see Figure 1). Where appropriate, if the nucleic acid analyte is complementary to the existing probe sequence, at least a portion of at least a double strand of hybridization complex is formed at the probe (see Figure 1). According to the present invention, the step of initiating the enzyme (see Figure 1C) is such that after the complex is formed, the action of the enzyme results in a measurable change in the mass of the complex. For example, if a hybridization complex comprises a shorter DNA probe and a relatively longer nucleic acid analyte is present, under suitable conditions and containing four nucleotide triphosphates (A; T; G; C), a polymerase can be used which can fill (at least in part) the single-stranded region 1310406 domain caused by the longer nucleic acid analyte (see Figure 1C and ID). This continuous polymerization occurs within a few minutes, based on the assumed average rate of association, i.e., the size of one base per minute. This example assumes that the two hybridized portions have different lengths and can be applied in the opposite example, that is, the length of the analyte is shorter than that of the probe. In this case, it is advantageous to design the probe to have a length of at least 100 nucleotides, and a single-strand region of the hybrid complex is expected, and it can be prolonged by the activity of the polymerase. Full, as close as possible to the surface of the sensor. This advantage can also be achieved if the probe is fixed by the 3' end and the single-stranded region is full in the solid phase. These beneficial specific examples can be transferred to not only polymerases, but also all enzymes which are generally suitable in accordance with the present invention, and which are readily accomplished by those skilled in the art, depending on the field in which they are intended to be applied. When there is no analyte in the sample to be tested that is complementary to the probe sequence, there is no hybridization complex at this time point due to different binding energies, so the next enzyme reaction does not occur, and there is no Enzyme-dependent measurement data can be recorded. Since the hybridization reaction which does not apply an electric field usually takes several hours, according to the present invention, the detection time is considerably reduced, so that it can occur in a short time window, and is more suitable for reading of the sensor. The polymerase activity as exemplified above can produce pyrophosphate ions which are released during the polymerization of the nucleotide triphosphate in the single-strand region of the hybrid complex and result in local acidification, thereby lowering the acid-base enthalpy . By partially arranging the pH sensor or pH detector (for example, acid-base sensing field effect transistor element (PH-ISFET)), if you want to detect the corresponding location of the factory 4«·:
L S 11 1310406 處酸鹼値的變化（見圖2 )。 進一步，本發明亦提供間接偵測聚合反應或結合反應 過程中釋放出來的焦磷酸根離子，也就是說，藉由一個二 級酵素系列反應。舉例來說，ATP硫化酶及腺苷-5’-磷酸化 硫（APS )參與此二級反應的第一步。在此例子中，ATP硫 化酶轉換在聚合反應或結合反應過程中合倂核苷酸所釋放 的焦磷酸根離子ΡΡι及APS變爲ATP。此產生的ATP接著 催化進一步的酶的反應，其係可被實際偵測到的。舉例來 說，此形成的ATP可催化螢光酶轉換螢光素的反應，導致 其發光而被本發明之光學感應器掃描到。 本發明方法之修飾例子是有關裝載與磁性球體一同使 用的核守酸三磷酸（Baselt，/oc. C2Y·，1998 )或與金屬粒子 一同使用（臨床微感應器（Clinical Micro-sensors)，c/Λ ，2000 )。此裝載的結果，由於與核苷酸三磷酸結合的固 體物之附加性質，使讀取的結果進一步加強。進而，核苷 酸三磷酸上會呈現顏色（染料/色素），且藉由固定的發光 二極體讀取。 因此，根據較佳的具體實例，感應器（至少一個）係選自 由包括電極構造、場效電晶體、磁感器、光感器及酸鹼感 應器所組成的群組，對應於完全的光譜使用。 更佳的具體例是有關於結合使用前述不同形式的感應 器。舉例來說，其可能可以最適化訊號強度或靈敏度，如 此可得到一個想要偵測的事件的可信度，如果此偵測的儀 器是適合於本發明之方法，其不只有一個探針專一的感應 12 1310406 器（例如場效電晶體）’亦包括其他形式的探針專一感應器（ 例如酸鹼感測場效電晶體元件（PH-ISFET))。必要時，這 些從多參數的測量所得到的數據，可使酵素依賴的訊號被 更正確的評估。 進而，此感應器可裝設一加熱元件。此類元件包括， 例如，在影像感應（CMOS)過程中應用的傳導體軌跡，後 來被下一層所覆蓋。如此有助於溫度循環的進行，舉例來 說，對文中本發明方法的PCR支援應用是令人滿意的。 因爲有感應器之被測物的加入脈衝，會產生所謂的加 成性波峰，最好是連續的操作本發明之方法（以直通模式）。 根據其他方面，提供一儀器以達成本發明之方法。 此儀器包含至少一固相、至少一核酸探針直接或間接 固定在其上，及至少一個感應器以偵測物理測量數據，此 處的感應器是此固相上不可缺少的一部分，且較佳係選自 於上文所定義之群組，包括電極構造、場效電晶體、磁感 器、光感器及酸鹼感應器。 根據本發明所提，較適合的儀器是，多數不同的核酸 探針以格子狀排列成微矩陣，此微矩陣更佳係提供至少一 個感應器於每一個固定的核酸探針處或每一個專一的偵測 區域。 由ΕΡ-Α-0 881 490得知的測量儀器，可測量至少一個 待測活細胞的特定物理或型態上的參數，經適當修改後’ 可根據本發明使用。此裝備如已述的含有多個感應器’此 感應器是所提供的儀器中不可缺少的部分，所欲檢測的物 13 1310406 質是固定在其上。 根據本發明，此儀器的輔助元件主要包括含嵌入之偵 測層的半導體材料，最好包含數個偵測器，其中至少一個 上述的感應器係倂入作爲偵測器，視需要結合之（見上述 )。進而，此輔助元件中可含有加熱的元件，以在實施過 程中提供不同的溫度（見上述）。在一更佳的具體實例中 ，此訊號的處理至少部份在所使用的感應晶片發生。 根據本發明之一個方面，被掃描輸出的測量數據，舉 例來說，可以利用相似的區域，直接在晶片上估計，例如 ，以每毫秒紀錄一數値，然後與先前測量而得的參考値做 比較，此參考値亦儲存在晶片內。此外，此操作模式使一 些非專一的干擾訊號，例如，分散向內的外部訊號，被計 算出來。 此感應器的表面設計，如微矩陣的排列方式，其上有 多數偵測區域需要被估計，此測量範圍或測量點的訊號可 依序偵測，例如，測量整排或整行的感應器表面，或分部 份測量（多重應用）。 舉例來說，經由類似的數字轉換後，從偵測儀所輸出 的電子訊號，可利用合適的線路，傳送至外部的評估儀器 (見上述）。 爲了使本發明之方法能運用在感應器的這一層，根據 另一個較佳的具體實例，可在其上覆蓋一層可以相結合的 物質。典型的是將感應器晶片表面，例如，這些含有二氧 化矽的組成，浸泡於一含有雙功能分子（所謂的連接物） 14 1310406 的溶液中，其具有鹵矽烷（例如，氯矽烷）或矽烷氧基， 可與此支持面連接，因此形成自行組合的單一層（SAM) ，經由感應器表面與受體之間產生共價鍵結。舉例來說， 此覆蓋可使用甘油三矽烷乙氧完成，例如，將其浸泡在含 有1%矽甲烷的甲苯溶液中，緩慢取回，置於120°C中烘烤 ，將其固定。一般利用這種方式覆蓋的厚度約有幾埃。連 接物與受體分子間經由另一個合適的功能基連接，例如， 胺基或環氧基。可連接多個不同的受體分子的適合雙功能 連接物，尤其是源於生物體的受體分子，使其接合到晶片 的表面，熟於此技術的人皆了解，較之於G.T. Hermans on， Academic Press 1996之“生物結合技術”。其可讓此接合的 物質變成一薄的聚合層，而產生具有功能的表面，參見WO 00/43539 。 根據本發明，前述做爲探針的核酸，可利用一般打印 的設備加以應用且固定。 舉例來說，DNA的雜交可利用本方法在晶片表面上建 立的方法實施。此可藉由，例如，PCR方法製備。在雜交 過程中，DNA被測物與感應器表面探針（如果存在）的配 對股結合。陽性雜交事件可利用本發明之方法偵測到。 特定位置質量增加的測量可用，例如，物理方法。舉 例來說，特定位置折射指數的改變、特定位置電阻或電子 傳導性的改變、特定位置光密度的改變，或甚至特定位置 二向色效應等，皆可被測量。 基本上’本發明普遍的方法適合廣大光譜區域的應用 15 1310406 ，其中在此區域內可區別待測樣品中特定被測物完整診斷 的偵測，與另一方面，利用本方法由複合物衍生的修飾來 測定序列數據，或是有關基因組功能關係的資訊。然而， 這種區別是供做舉例說明的目的，基本上，本發明公開的 適用性，不限於此。 舉例來說，本發明之方法尤其適用於測定可經由巢式 PCR平行放大而產生的DNA的序列，較佳係於一結合液相/ 固相的DNA微矩陣系統，因爲其不需使用修飾的核苷酸（ 例如生物素或毛地黃素），以及螢光染料和其他傳統使用的 標記物質。在結合液相/固相的DNA微矩陣系統的巢式PCR (見圖4)具有與傳統PCR相同的敏感度，也就是說，在 液相中進行的反應，但同時確保比傳統雜交分析及引子延 展分析更高的專一性。此優點是由於引子/樣品DNA/聚合 酶系統固有的關於放大反應之專一性被大大的甚至更進一 步增加，其係藉由固定在固體撐體的內部PCR引子（因此也 做爲探針用）及複製子（amplkon)間的專一交互作用。整個結 果，其專一性是優於，例如，5’-外切核酸酶分析（例如， 當使用TaqManTM聚合酶時）。 從感應器發出的訊號係利用紀錄器紀錄。紀錄器具有 一快速轉換器，將相似的偵測訊號轉換成數値，儲存在記 憶中。其較佳係用於評估即時產生的訊號，但也可在時間 延遲後評估。普通的微處理器可用來評估此數値。 【實施方式】 本發明舉下列實施例詳述。The change in acid-base enthalpy at L S 11 1310406 (see Figure 2). Further, the present invention also provides for indirect detection of pyrophosphate ions released during polymerization or binding reactions, that is, by a series of secondary enzyme reactions. For example, ATP sulfide and adenosine-5'-phosphorylated sulfur (APS) are involved in the first step of this secondary reaction. In this example, the ATP-sulfurase converts the pyrophosphate ions and APS released by the hydrazine nucleotides into ATP during the polymerization or binding reaction. The resulting ATP then catalyzes a further enzyme reaction which is actually detectable. For example, the ATP formed can catalyze the reaction of the luciferase to convert luciferin, causing it to illuminate and be scanned by the optical sensor of the present invention. A modified example of the method of the present invention relates to loading a phosphoric acid triphosphate (Baselt, /oc. C2Y., 1998) for use with a magnetic sphere or for use with a metal particle (Clinical Micro-sensors, c /Λ, 2000). As a result of this loading, the results of the reading are further enhanced by the additional nature of the solids bound to the nucleotide triphosphate. Further, a color (dye/dye) appears on the nucleotide triphosphate and is read by a fixed light-emitting diode. Therefore, according to a preferred embodiment, the inductor (at least one) is selected from the group consisting of an electrode configuration, a field effect transistor, a magnetic sensor, a photosensor, and an acid-base sensor, corresponding to a complete spectrum. use. A more specific embodiment is related to the use of the aforementioned different forms of inductors. For example, it may be possible to optimize the signal strength or sensitivity so that the confidence of an event to be detected is obtained. If the detected instrument is suitable for the method of the present invention, it is not only one probe specific. The sensing 12 1310406 device (eg field-effect transistor) also includes other forms of probe-specific sensors (eg acid-base sensing field effect transistor elements (PH-ISFETs)). These data from multi-parameter measurements can be used to more accurately evaluate enzyme-dependent signals when necessary. Furthermore, the inductor can be provided with a heating element. Such components include, for example, conductor traces applied during image sensing (CMOS) processes, which are then covered by the next layer. This facilitates the progress of the temperature cycle, for example, the PCR support application of the method of the invention herein is satisfactory. Because of the pulse of the addition of the object of the sensor, a so-called additive peak is produced, preferably by continuously operating the method of the invention (in pass-through mode). According to other aspects, an apparatus is provided to achieve the method of the present invention. The apparatus comprises at least one solid phase, at least one nucleic acid probe directly or indirectly immobilized thereon, and at least one sensor for detecting physical measurement data, wherein the sensor is an indispensable part of the solid phase, and Preferably, the group is selected from the group defined above, including electrode structures, field effect transistors, magnetic sensors, light sensors, and acid-base sensors. According to the present invention, a more suitable instrument is that a plurality of different nucleic acid probes are arranged in a lattice form into a micromatrix, and the micromatrix preferably provides at least one sensor at each fixed nucleic acid probe or each specific one. Detection area. A measuring instrument known from ΕΡ-Α-0 881 490, which measures at least one specific physical or type parameter of a living cell to be tested, can be used according to the invention, as appropriate. This equipment contains a plurality of inductors as already described. This sensor is an indispensable part of the instrument provided, and the substance 13 1310406 to be tested is fixed thereto. According to the invention, the auxiliary component of the apparatus mainly comprises a semiconductor material comprising an embedded detection layer, preferably comprising a plurality of detectors, wherein at least one of the above-mentioned inductors is incorporated as a detector, if necessary, See above). Further, the auxiliary element may contain heated elements to provide different temperatures during implementation (see above). In a more preferred embodiment, the processing of this signal occurs at least in part on the sensing wafer used. According to one aspect of the invention, the measured data that is scanned for output, for example, can be estimated directly on the wafer using similar regions, for example, by recording a number of frames per millisecond, and then with the previously measured reference frame. In comparison, this reference 値 is also stored in the wafer. In addition, this mode of operation allows some non-specific interference signals, such as external signals that are scattered inward, to be calculated. The surface design of the sensor, such as the arrangement of the micro-matrix, on which many detection areas need to be estimated, the measurement range or the measurement point signal can be detected sequentially, for example, measuring the entire row or the entire line of sensors Surface, or partial measurement (multiple applications). For example, after a similar digital conversion, the electronic signal output from the detector can be transmitted to an external evaluation instrument using a suitable line (see above). In order to enable the method of the present invention to be applied to this layer of the inductor, according to another preferred embodiment, a layer of material that can be combined can be applied thereto. Typically, the surface of the sensor wafer, for example, the composition containing cerium oxide, is immersed in a solution containing a bifunctional molecule (so-called linker) 14 1310406 having a halodecane (e.g., chlorodecane) or decane. The oxy group, which can be attached to this support surface, thus forms a self-assembled single layer (SAM) that creates a covalent bond between the surface and the acceptor via the sensor. For example, the coverage can be accomplished using glycerol trioxane ethoxylate, for example, by soaking it in a toluene solution containing 1% hydrazine methane, slowly withdrawing it, baking at 120 ° C, and fixing it. Generally, the thickness covered by this method is about several angstroms. The linker is linked to the acceptor molecule via another suitable functional group, for example, an amine group or an epoxy group. Suitable bifunctional linkers that can link a plurality of different receptor molecules, particularly receptor molecules derived from organisms, are bonded to the surface of the wafer, as is known to those skilled in the art, as compared to GT Hermans on , Academic Press 1996, "Bio-binding technology." It allows the bonded material to become a thin polymeric layer to produce a functional surface, see WO 00/43539. According to the present invention, the aforementioned nucleic acid as a probe can be applied and fixed by a general printing apparatus. For example, hybridization of DNA can be carried out by the method of establishing the method on the surface of the wafer. This can be prepared, for example, by a PCR method. During the hybridization process, the DNA analyte is bound to the aligned strand of the sensor surface probe (if present). Positive hybridization events can be detected using the methods of the invention. Measurements of increased mass at a particular location may be used, for example, physical methods. For example, changes in refractive index at a particular location, changes in specific positional resistance or electron conductivity, changes in optical density at a particular location, or even a specific position dichroic effect can be measured. Basically, the general method of the present invention is suitable for the application of a wide spectral region 15 1310406, in which the detection of a complete diagnosis of a specific test substance in a sample to be tested can be distinguished, and on the other hand, the method is derived from the complex using the method. Modifications to determine sequence data or information about genomic functional relationships. However, such a distinction is for illustrative purposes, and basically, the applicability of the present disclosure is not limited thereto. For example, the method of the present invention is particularly useful for determining the sequence of DNA that can be produced by nested PCR parallel amplification, preferably in a liquid/solid phase DNA micromatrix system, since it does not require modification. Nucleotides (such as biotin or digoxigenin), as well as fluorescent dyes and other commonly used labeling substances. Nested PCR (see Figure 4) in a DNA micro-matrix system combined with a liquid/solid phase has the same sensitivity as conventional PCR, that is, the reaction carried out in the liquid phase, but at the same time ensures better than conventional hybridization analysis. The primer extension analyzes the higher specificity. This advantage is due to the fact that the specificity of the amplification reaction inherent in the primer/sample DNA/polymerase system is greatly and even further increased by the internal PCR primer immobilized on the solid support (and therefore also as a probe). And the specific interaction between the amplicons. The overall result is superior to, for example, 5'-exonuclease assays (e. g., when using TaqManTM polymerase). The signal from the sensor is recorded using a recorder. The recorder has a fast converter that converts similar detection signals into numbers and stores them in memory. It is preferably used to evaluate signals that are generated on the fly, but can also be evaluated after a time delay. A normal microprocessor can be used to evaluate this number. [Embodiment] The present invention is described in detail in the following examples.
16 1310406 根據本發明製備一感應晶片 CMOS感應器係使用1.2微米的CMOS方式裝設在一 5 英吋或6英吋的膠紙上。每一個場效電晶體被置於P-受質 上的η槽。藉由引流器及來源區域的輸入，產生場的氧化 作用。應用一厚度大約10 nm的熱閘門氧化物。此閘門在 接下來的反應過程中，被聚矽化合物保護。利用化學氣相 沉積步驟（CVD )加上一層二氧化矽，鋁也噴灑於其上。 鈍化係藉氮化矽沉積層及氧化矽沉積層達成。此絕緣閘門 暴露在接下來的蝕刻步驟中。 覆蓋CMOS感應器 上述CMOS感應器的製備，是浸泡在含有1% G0PS及 含0.1%三乙胺的甲苯溶液中大約2小時使覆蓋上矽甲烷。 接著從溶液中取出晶片，待一段時間滴乾之後，置於乾燥 箱中，以120°C固定大約2小時。 必要時，以這種方式覆蓋完成的晶片可以儲存在乾燥 的環境中，直到進行生物連接反應爲止。 宴核苷酸探針生物連接反應 上述覆蓋完成的晶片，利用傳統技術，以非接觸的步 驟加上5’-胺基修飾的寡核苷酸探針。爲了達到此目的，此 寡核苷酸保存在濃度爲5 μΜ PBS的緩衝溶液中。待完成 (printing)之後，此連接反應繼續在一 50°C的潮濕容器中進 行。最後，此晶片以蒸餾水沖洗，再利用甲醇沖洗使其乾 燥。任何殘留的溶劑，最後置於排氣櫃中，以蒸發作用移 除。 i ^ } 17 1310406 ' 製備樣品的步驟 來自人類DNA分離物的血色素基因片段藉由PCR放大 。在放大反應中使用合適的引子序列，例如，美國專利 5,712,098 中所述。 此反應的混合物包括以下的標準試劑（引子：0.5 μΜ ;dATP、dCTP、dGTP : 0.1 mM ; dTTP 0.08 mM ; PCR 緩衝 液，MgCh : 4 mM ; HotStarTaq ( Perkin Elmer) 2 單位（50 μΐ))。原本的核苷酸在PCR反應過程（35個循環，95°C 5 分鐘、95t 30 秒、60°C 30 秒、72t 30 秒、72t 7 分 鐘）中倂入新合成的DNA中。接著，加入T7 Gen6-外切核 酸酶（每一次PCR，100單位/50 μΐ)，以產生單股DNA， 且加熱此反應（37°C 30分鐘，85°C 10分鐘）。 雜交反應 上所述的反應，在5倍SSPE，0.1% SDS ( 12 μΐ)緩衝 溶液中，在晶片上進行雜交，在50°C潮濕的容器中大約2 小時。最後，以2倍SSPE，0.1% SDS沖洗，再以水沖洗 乾淨晶片。 【圖式簡單說明】 (一）圖式部分 本發明及有益的具體實例將以圖輔助詳述： 圖1說明本發明方法之一具體實例的步驟的圖解。（A )核酸探針⑵共價鍵結至表面。（B )加入核酸被測物（1) 之後，形成雜交複合物，通常需要數小時。（C)使用一合 適的酵素，例如，聚合酶，且同時具有四種核苷酸A、T、 1310406 G及C (以DNA爲例），此複合物的單股區域，大約在幾 分鐘的短時間內被塡滿，（D)因此很快的產生一個訊號， 此訊號會被感應器（4)讀取，其是此固相不可缺的一部分。 圖2說明使用一適合的聚合酶反應的較佳具體實例之 原理，其會釋放出焦磷酸根離子（5)，導致局部酸鹼値改變 。此改變可被固定的感應器（4)偵測到。 圖3說明本文中影像感應（CMOS)過程建構的場效電 晶體。此場效電晶體包含，舉例來說，在其η槽中具有一 層ρ-η-ρ(6)的構造，其表面有一層薄的絕緣體（10)(例如， 10 run的熱氧化物），其上可直接或間接供探針的應用，以 進行雜交反應。在較佳的具體實例中，場效電晶體區域所 刻劃的保護（7)是鈾刻成鋒利的或刻度的方式，因此，雜交 及質量增加（8)的過程可在凹陷處進行。此儀器的表面藉使 用（例如）貴金屬或疏水/親水性物質（9)，可對於核酸分子的 雜交產生主動或被動的影響力，。在測量溶液（11)中，例如 ，1M碳酸氫鈉，在閘門處測量電介質性質的改變，其係由 於雜交複合物的單股區域塡滿。因此造成的高原電位轉變 ，可使用位於溶液中的參考電極（12)，藉由場效電晶體讀取 。舉例來說，引流器與源頭之間的電流，或參考電極與源 頭間的電壓，皆可被紀錄成訊號（見，例如，B. Palan等人 ’ Fundamental Noise Limits of ISFET-based Microsystems， Poster contribution 4P26 5 EUROSENSORS XIII ( ISBN 90-76699-02-X) ，p. 169 ff.，1999 )。 圖4說明平行放大的步驟，基於所知的“巢式晶片16 1310406 Preparation of a Sensing Wafer in Accordance with the Invention A CMOS sensor is mounted on a 5 inch or 6 inch adhesive tape using a 1.2 micron CMOS. Each field effect transistor is placed in the n-well on the P-substrate. The oxidation of the field is produced by the input of the drain and the source region. Apply a thermal gate oxide approximately 10 nm thick. This gate is protected by a polyfluorene compound during the subsequent reaction. A layer of cerium oxide is applied by a chemical vapor deposition step (CVD), and aluminum is also sprayed thereon. Passivation is achieved by a tantalum nitride layer and a yttrium oxide layer. This insulating gate is exposed to the next etching step. Overlay CMOS sensor The above CMOS sensor was prepared by immersing it in a toluene solution containing 1% G0PS and 0.1% triethylamine for about 2 hours to cover the methane. The wafer was then taken out of the solution, and after being dried for a while, it was placed in a dry box and fixed at 120 ° C for about 2 hours. If necessary, the wafers covered in this manner can be stored in a dry environment until a bio-ligation reaction is performed. Feast nucleotide probe bioligand reaction The above covered wafers were grafted with a 5'-amino modified oligonucleotide probe in a non-contact procedure using conventional techniques. To achieve this, the oligonucleotide was stored in a buffer solution at a concentration of 5 μM PBS. After the printing, the ligation reaction was continued in a humid container at 50 °C. Finally, the wafer was rinsed with distilled water and rinsed with methanol to dry. Any residual solvent is finally placed in the venting cabinet and removed by evaporation. i ^ } 17 1310406 'Step of preparing the sample The hemoglobin gene fragment from the human DNA isolate was amplified by PCR. A suitable primer sequence is used in the amplification reaction, for example, as described in U.S. Patent 5,712,098. The mixture of this reaction included the following standard reagents (primer: 0.5 μΜ; dATP, dCTP, dGTP: 0.1 mM; dTTP 0.08 mM; PCR buffer, MgCh: 4 mM; HotStarTaq (Perkin Elmer) 2 units (50 μM)). The original nucleotide was cleaved into the newly synthesized DNA during the PCR reaction (35 cycles, 95 ° C for 5 minutes, 95 t 30 seconds, 60 ° C for 30 seconds, 72 t 30 seconds, 72 t 7 minutes). Next, T7 Gen6-exonuclease (100 units/50 μM per PCR) was added to generate a single strand of DNA, and the reaction was heated (37 ° C for 30 minutes, 85 ° C for 10 minutes). Hybridization reactions The reactions described above were hybridized on a wafer in a 5x SSPE, 0.1% SDS (12 μM) buffer solution for approximately 2 hours in a humidified container at 50 °C. Finally, rinse with 2 times SSPE, 0.1% SDS, and rinse the wafer with water. BRIEF DESCRIPTION OF THE DRAWINGS (I) Schematic Part The present invention and advantageous specific examples will be described in detail with reference to the accompanying drawings: Figure 1 illustrates an illustration of the steps of a specific example of the method of the present invention. (A) The nucleic acid probe (2) is covalently bonded to the surface. (B) After the addition of the nucleic acid test substance (1), a hybridization complex is formed, which usually takes several hours. (C) using a suitable enzyme, such as a polymerase, and having four nucleotides A, T, 1310406 G, and C (in the case of DNA), a single-stranded region of the complex, in about a few minutes When it is full in a short time, (D) will soon generate a signal, which will be read by the sensor (4), which is an indispensable part of this solid phase. Figure 2 illustrates the principle of a preferred embodiment using a suitable polymerase reaction which releases pyrophosphate ions (5) resulting in a local acid-base enthalpy change. This change can be detected by the fixed sensor (4). Figure 3 illustrates a field effect transistor constructed by an image sensing (CMOS) process herein. The field effect transistor includes, for example, a structure having a layer of ρ-η-ρ(6) in its n-groove having a thin insulator (10) on its surface (for example, 10 run of thermal oxide), The probe can be applied directly or indirectly to carry out the hybridization reaction. In a preferred embodiment, the characterization of the field effect transistor region (7) is that the uranium is engraved in a sharp or graduated manner, so that the process of hybridization and mass increase (8) can be performed in the depression. The surface of the instrument, by means of, for example, a noble metal or a hydrophobic/hydrophilic substance (9), can have an active or passive influence on the hybridization of the nucleic acid molecule. In the measurement solution (11), for example, 1 M sodium hydrogencarbonate, the change in dielectric properties was measured at the gate due to the fullness of the single-strand region of the hybridization complex. The resulting plateau potential transition can be read by a field effect transistor using a reference electrode (12) located in solution. For example, the current between the drain and the source, or the voltage between the reference electrode and the source, can be recorded as a signal (see, for example, B. Palan et al.' Fundamental Noise Limits of ISFET-based Microsystems, Poster contribution 4P26 5 EUROSENSORS XIII ( ISBN 90-76699-02-X), p. 169 ff., 1999). Figure 4 illustrates the steps of parallel amplification, based on known "nested wafers"
19 1310406 (Nested On Chip)”PCR ( NOC，見上述），偕同 FET 掃描電 位變化。在整個NOC過程中，一個探針處的電位變化示於 A。X軸代表循環次數，Y軸代表測量的電壓。引子分子（ 相當於探針分子）連接到探針的位置，標示於X軸下方： 剛開始的循環中，只有一些引子被延長，接著，進入中間 的循環，以指數的形式大量的增加，在後段循環中，逐漸 增加成飽和狀態（大部分的引子已經延長）。此曲線顯示 當探針位置的質量增加時，所測得電壓上升的情形。B顯 示單一循環（循環次數在中間）的電位圖形。除了引子， 模板及引子從左到右的延長，在此皆有顯示。此說明顯示 ，電壓的上升是引子延長的函數。 (二）元件代表符號 1 :核酸被測物 2 :核酸探針 3:四種核苷酸三磷酸（A; T; G; C) 4 :感應器 5:焦磷酸根離子 6 : p-n-p 層 7:場效電晶體區域所刻劃的保護 8 :雜交及質量增加 9 :貴金屬或疏水/親水性物質 10 :絕緣體 11 :待測溶液 12 :參考電極19 1310406 (Nested On Chip) PCR (NOC, see above), with the FET scanning potential change. During the entire NOC process, the potential change at one probe is shown in A. The X axis represents the number of cycles, and the Y axis represents the measurement. Voltage. The position of the primer molecule (equivalent to the probe molecule) attached to the probe, indicated below the X axis: In the beginning of the cycle, only some of the primers are extended, and then, into the middle of the cycle, a large increase in exponential form In the latter cycle, it gradually increases to saturation (most of the primers have been extended.) This curve shows the measured voltage rise when the mass of the probe position increases. B shows a single cycle (the number of cycles is in the middle) The potential pattern. In addition to the primer, the template and the extension of the primer from left to right are shown here. This description shows that the voltage rise is a function of the extension of the primer. (2) Component Representation Symbol 1: Nucleic Acid Detector 2: Nucleic acid probe 3: four nucleotides triphosphate (A; T; G; C) 4 : sensor 5: pyrophosphate ion 6 : pnp layer 7: field effect transistor region characterization protection 8 : hybridization Mass increased by 9: noble metal or hydrophobic / hydrophilic substance 10: insulator 11: 12 test solution: a reference electrode